1. Field of the Invention
The present invention relates to cells or plants having the ability of producing polylactate or its copolymers and a method for preparing polylactate or its copolymers using the same, more specifically, relates to cells or plants capable of expressing a gene encoding an enzyme converting lactate into lactyl-CoA and a gene encoding an enzyme involved in PHA synthesis and a method for preparing polylactate or hydroxyalkanoate-lactate copolymer, the method comprises: culturing the cells in a medium containing lactate, carbon sources providing hydroxyalkanoyl-CoA or lactate and various hydroxyalkanoate, or culturing the plants.
2. Description of the Background Art
Polylactate (PLA) is a typical biodegradable polymer originated from lactate, which has a variety of applications as a common or a medical polymer. At present, PLA is being prepared by polymerizing lactate which is produced by fermenting microorganisms, but only low molecular weight PLA (1000-5000 dalton) is produced by direct polymerization of lactate. To synthesize high molecular weight (>100,000 dalton) of PLA, a method polymerizing low molecular weight PLA obtained by direct polymerization of lactate using a chain coupling agent to obtain higher molecular weight PLA can be used. But it has disadvantages in that, the process for preparing PLA of high molecular weight is complicated due to the addition of a solvent or a chain coupling agent and also it isn't easy to remove them. At present, in the process for preparing commercially available PLA of high molecular weight, a method, in which lactate is converted into lactide to synthesize PLA by cyclodehydration of the lactide ring, is being used.
Meanwhile, PHA is a polyester which microorganisms accumulate therein as a carbon and energy storage compound when other nutritive elements, for example, phosphorus, nitrogen, magnesium, oxygen, are deficient while the carbon source is in excess. PHA is recognized as an alternative material for existing synthesized plastics since it has similar properties to synthetic polymers originating from petroleum, and, at the same time, shows an excellent biodegradation rate.
The existing PHA is divided into SCL-PHA(short-chain-length PHA) having short carbon chains and MCL-PHA(medium-chain-length PHA) having long carbon chains. A gene synthesizing PHA was cloned from Ralstonia eutropha, Pseudomonas and, PHA consisting of various monomers was synthesized by recombinant microorganisms (Qi et al., FEMS Microbiol. Lett., 157:155, 1997; Qi et al., FEMS Microbiol. Lett., 167:89, 1998; Langenbach et al., FEMS Microbiol. Lett., 150:303, 1997; WO 01/55436; U.S. Pat. No. 6,143,952; WO 98/54329; WO 99/61624).
To produce PHA in microorganisms, an enzyme which converts metabolites into a PHA monomer and PHA synthase which synthesizes the PHA polymer using the PHA monomers are required. PHA synthase synthesizes PHA using hydroxyacyl-CoA as a substrate and β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), cloned from Ralstonia eutropha etc., 3-hydroxydecanoyl-ACP:CoA transferase (PhaG) cloned from Pseudomonas, (R)-specific enoyl-CoA hydratase (PhaJ) derived from Aeromonas caviae and Pseudomonas aeruginosa (Fukui et al., J. Bacteriol., 180:667, 1998; Tsage et al., FEMS Microbiol. Lett., 184:193, 2000), 3-ketoacyl-ACP reductase (FabG) derived from E. coli and Pseudomonas aeruginosa (Taguchi et al., FEMS Microbiol. Lett., 176:183, 1999; Ren et al., J. Bacteriol., 182:2978, 2000; Park et al., FEMS Microbiol. Lett., 214:217, 2002) are known as enzymes capable of generating hydroxyacyl-CoA which is a substrate of PHA. Various kinds of PHAs have been synthesized with these enzymes using hydroxyalkanoates hydroxylated at various positions in the carbon chain (mainly the 3, 4, 5, and 6 positions). However, it has been reported that it has little PHA synthase activity on hydroxyalkanoate which is hydroxylated at the 2-position (Zhang et al., Appl. Microbiol. Biotechnol., 56:131, 2001; Valentin and Steinbuchel, Appl. Microbiol. Biotechnol., 40:699, 1994; Yuan et al., Arch. Biochem. Biophyics., 394:87, 2001). Thus far, there have been reports of PHA synthase activity on lactyl-CoA measured in vitro as PHA polymerase activity on lactyl-CoA. However, but PHA synthase activity on lactyl-CoA is very weak (Zhang et al., Appl. Microbiol. Biotechnol., 56:131, 2001; Valentin and Steinbuchel, Appl. Microbiol. Biotechnol., 40:699, 1994). That is, there are no examples of natural production or production by recombinant cells of PHA and its copolymers because a hydroalkanoate, such as lactate hydroxylated at the 2-carbon position, is not a suitable substrate for PHA synthase.
Therefore, the present inventors have expended extensive efforts to produce high molecular weight PLA and its copolymers using cells or plants. As disclosed herein, they have discovered that that poly[hydroxyalkanoate-co-lactate] copolymers is produced by culturing recombinant Ralstonia eutropha transformed with the propionyl-CoA transferase gene (pct) derived from Clostridium propionicum which is a gene encoding an enzyme that converts lactate into lactyl-CoA in a production medium containing lactate. PLA was produced by culturing in a production medium containing lactate the recombinant E. coli transformed with the PHA synthase gene derived from Bacillus cereus and the pct gene.